a. Field of the Invention.
The invention relates to the field of the fluorescent detection of small quantities of molecules in the presence of background or competing fluorescence. In particular, the invention relates to a new fluorescent antibody, and a method which utilizes the unique fluorescent properties of the new fluorescent antibody to minimize the effect of background fluorescence thereby greatly increasing the effective sensitivity of fluorescent detection methods.
b. Prior Art.
The fluorescent antibody (FAB) technique was originated by Coons and co-workers in 1941. The original concept was to couple a dye to an antibody which could then bind to an antigen. In its initial forms it was primarily used to visualize or trace the distribution of certain antigens in tissues or on cells. The use of a fluorescent dye, in contrast with ordinary non-fluorescing dyes which had been previously used as markers, resulted in a thousand fold increase in sensitivity.
In the years that followed, the techniques of conjugating fluorescent dyes were improved and more stable dyes were synthesized. In 1950 and 1951 Coons and co-workers developed improved methods for synthesizing fluorescein isocyanate and conjugating it with antibodies. This, coupled with the development of readily available fluorescent microscopes, placed the FAB methodology on firm footing and paved the way for its extended use in medicine.
In the late 1950's three other important stable fluorescent dyes were developed, Rhodamine B200, 1-dimethylaminonaphthalene 5-sulfonyl chloride (DANS), and fluorescein isothiocyanate (FITC). To this date, however, the most commonly used dye in FAB work is FITC.
Until quite recently the FAB technique has been used primarily as a tracer, for visualization of the location of antigens on a sample using a fluorescent microscope. These uses were not quantitative; the fluorescence of the FAB was used only to indicate the presence or absence of a particular antigen or its distribution on tissue or a cell.
Presently, a considerable amount of effort is being expended to develop immunological methods which would enable the quantitative measurement of specific antigens in the blood or other body fluids. These antigens may be naturally occurring substances such as cancer antigens, renin, or thyroxine or administered drugs such as digoxin, or methotrexate.
One immunological test which has been successfully applied to assays requiring high sensitivity is radio-immunoassay (RIA). In RIA, antigen of the type to be assayed is labeled radioactively, allowed to compete for available antibodies with antigen to be assayed and after suitable procedures, radioactivity is measured and the amount of antigen present deduced. Though RIA is sufficiently sensitive for most subtle assays, it suffers from disadvantages because of the radioactive label. These disadvantages include extra care in handling radioactive material, the problem of disposal of radioactive wastes and the finite shelf life of radioactive antigens, typically 60 days.
The possibility of utilizing FAB instead of radioactively labeled antigens in immunoassay has been considered by workers in the field because of the extreme sensitivity of the fluorescence method, especially using laser excitation. However, the presence of background fluorescence of sample holders, reagents, administered drugs and organic material in the blood serum or urine has interfered with the attainment of the high sensitivity needed for immunofluorometric assay.
One method that has been suggested to increase sensitivity in immunofluorometric analysis is "mechanical amplification". This method utilizes microscopic balls labeled with fluorescent dyes and antibodies and is described in U.S. Pat. No. 3,853,987 issued to Dryer. The Dryer method attempts to increase the signal from the antigen-antibody complex.
In 1975, Wieder filed a co-pending patent application, Ser. No. 591,305, filed June 30, 1975 now U.S. Pat. No. 4,058,732, issued Nov. 15, 1977, in which he disclosed a method for improved fluorescent spectroscopy wherein target molecules whose presence is to be measured are combined with fluorescent molecules having a relatively long fluorescent decay lifetime. These tagged target molecules are excited with a pulse of ultraviolet or visible radiation and a detection system is gated on only after background fluorescence has substantially decayed, but while the fluorescent tag attached to the target molecules is still actively decaying. The amount of fluorescence tag emission which is measured is indicative of the target molecule content of the sample.
In that patent application, an apparatus for improved fluorescent spectrofluorometry was also disclosed which includes a pulsed excitation source and a gating means connected to a detection system for gating the detection system on after tagged target molecules have been excited and competing background fluorescence has substantially decayed. In an article in Analytical Chemistry 46, 1960 (1974) R. E. Brown et al. have disclosed a gated spectrofluorometric apparatus.
In Wieder's application two examples of a tagging agent were disclosed. One example was the class of rare-earth organo complexes, i.e. rare-earth chelates, which are non-specific target tags and have fluorescent lifetimes of 100 to 800 microseconds. The other example, was a FAB which consists of a target specific antibody conjugated with pyrenebutyrate. However, even though this FAB has a fluorescent lifetime of about 200 nanoseconds which is longer than expected background fluorescence, it limits the delay time before the detection apparatus is turned on, thus limiting the extent to which background fluorescence has decayed before the measurement begins.
An object of the invention is to devise a new FAB family with a longer decay lifetime than any prior FAB for use in an immunofluorometric assay and other applications.